Waterborne diseases of significant public-health impact over the past decade
have been caused by several microorganisms, including toxin-producing strains
of the fecal bacteria E. coli (Craun et al., 1997), and intestinal protozoan
parasites such as Giardia and Cryptosporidium (Kramer et al., 1993; Rose, 1997).
Such protozoans are readily spread through fecal contamination of water by the
protozoan cysts or oocysts. Moreover, cysts and oocysts excreted by animals
can infect humans. Any comprehensive examination of water quality should include
monitoring for microbial flora, with an emphasis on the pathogens of greatest
health concern. Current reservoir research at the University of Missouri - Columbia
has the overall objective of determining reservoir water quality within the
state (as measured by nutrients, water clarity and algal biomass), and quantifying
the major factors controlling present conditions (land use and climate). We
propose to expand this research to include measurements of pathogenic microorganisms
in surface waters.

Water is a critical resource in Missouri, in all states of the region, and
throughout the nation. The proposed research is essential for a complete understanding
of the factors that influence water quality, including the incidence and sources
of potential pathogens/parasites in water, and the sources of potential long-term
changes in water quality independent of climatic induced variations. Land use
practices within a given watershed will affect water quality; this is of particular
impact, with respect to waterborne pathogens, in regions where fecal contamination
of water by humans, domestic animals and/or wildlife is prevalent. This project
is viewed as an initial step towards developing and applying the techniques
necessary to conduct widespread monitoring for the occurrence of potential pathogenic
microorganisms, and to identify their source of origin in surface waters. As
such, this study will provide a model for future programs assessing microbiological
and chemical parameters of water quality in wetland treatment systems, environmental
wetlands, and drinking water and recreational reservoirs.

Results or Benefits of Research

This research will result in the development of molecular techniques for the
measurement of waterborne pathogens, and the ultimate application of that methodology
to studies of wetlands and reservoirs in Missouri. We will obtain data on the
incidence of total coliforms, fecal coliforms, toxin-producing strains of E.
coli, and the protozoan parasites Giardia and Cryptosporidium in the Eagle Bluffs
wildlife conservation area and the adjacent wetland treatment system for city
wastewater. Measurement of the bacteria and the protozoan cysts and oocysts
will be achieved using specific assays based on the polymerase chain reaction
(PCR). In addition, bacteria will be measured using standard bacteriological
techniques. Our results will allow us to determine the incidence of these microorganisms
in the waters surveyed throughout the year, the correlation between the molecular
methodology developed and the standard bacteriological methods, and the relationship
between the occurrence of these pathogens and other limnological parameters.
We will also determine whether the presence of the fecal contaminant E. coli
serves as a reliable indicator for the presence of Giardia cysts and/or Cryptosporidium
oocysts in these surface waters.

A focus on the nearby Eagle Bluffs region will allow us to refine our ability
to monitor for the presence of waterborne fecal bacteria and pathogenic protozoans,
and to relate such presence to other parameters of water quality and to land
use practices. It will also allow us to assess the efficacy of the wetland treatment
system with respect to reducing the density of these microorganisms. Results
will be important for the city of Columbia, and may serve as a model for other
constructed wetland treatment systems. While we will focus on Missouri waters,
the techniques we develop for monitoring waterborne fecal and pathogenic microorganisms
--- including the concepts involved in the data acquisition and data analysis
--- will be applicable to water quality monitoring elsewhere. Ultimately, we
expect that the molecular assays for biomonitoring developed in this project
will be incorporated as a regular part of all our programs for monitoring water
quality in Missouri reservoirs. This multi-disciplinary approach may serve as
a model for other monitoring programs. Moreover, the tools developed in this
project will position us for 'source tracking' of pathogens. The Eagle Bluffs
Conservation Area is of particular interest in this respect, given that human
wastewater treatment cells serve as a primary source of water for the conservation
wetlands, and that these wetlands provide habitat for many types of wildlife,
including mammals as well as large numbers of migratory waterfowl.

Nature, scope and objectives of the research

We plan to develop efficient and cost effective molecular techniques to measure
the presence of coliform bacteria and parasites in wetlands. One of these techniques
is based on the polymerase chain reaction (PCR), an extremely powerful enzymatic
reaction used to produce relatively large amounts of a specific DNA fragment
from vanishingly small amounts of initial genetic material. This molecular technique
is commonly used for the identification of organisms through "DNA fingerprinting",
and is often employed in forensics and medical diagnostics. We will develop
a sensitive, specific, reproducible and efficient PCR-based assay for the detection
and eventual quantitation of fecal coliforms, including toxic strains of Escherichi
coli. The sensitivity of the PCR-based approach will provide greatly enhanced
opportunities for the rapid identification of specific strains of microorganisms
from environmental water samples. Moreover, this technique will facilitate studies
by allowing for the long-term archiving of microorganisms collected from water
samples onto membrane filters and available for future molecular assays in the
laboratory. These studies will allow us to determine whether E. coli is a reliable
water quality surrogate for the parasitic protozoans Giardia and Cryptosporidium
in Missouri wetlands and reservoirs. They will also allow us to determine the
relationship between the presence of fecal and pathogenic microorganisms, and
other standard limnological parameters of water quality. As such, this project
is a natural extension of our study of factors influencing lake fertility. Simply
stated, our working hypothesis is that factors favoring the growth of algae
in lakes (nutrient inputs from farmland and urban/suburban areas in the catchments,
and certain climate patterns) may likewise correlate with the presence (abundance)
of pathogenic organisms.

Reservoir research at the University of Missouri - Columbia has the overall
objective of determining reservoir water quality within the state (as measured
by nutrients, water clarity and algal biomass), and quantifying the major factors
controlling present conditions (land use and climate). Ultimately, we will determine
whether our lake resources are stable or changing with time; where conditions
are degrading, we will recommend remedial action based on lake models specific
to conditions in our state. This regional study of lake eutrophication (enrichment)
assumes that excessive nutrient inputs to lakes produce changes that are detrimental
to their function and use. Past studies have resulted in publications on the
major features of our reservoirs and empirical models which describe composite
patterns in lake water quality. Work in progress includes using remote sensing
techniques to quantify influences of watershed characteristics on lake fertility.
Also, we are developing a model to distinguish long-term trends from short-term
fluctuations caused by climate.

With this proposal, we hope to expand our approach to include measurements
of fecal microorganisms, including pathogens. Numerous outbreaks of waterborne
disease in drinking water and recreational lakes, caused by microorganisms such
as toxin-producing strains of the fecal bacteria E. coli and the protozoan parasites
Giardia and Cryptosporidium, highlight the need for an understanding of the
incidence and sources of these microorganisms in surface waters. As obligate
intestinal parasites, such protozoans are present in feces as cysts or oocysts,
and are readily spread through fecal contamination. Cysts and oocysts excreted
by domestic animals can infect humans. Any comprehensive examination of water
quality should include monitoring for microbial flora with an emphasis on the
pathogens of greatest public health concern. Our approach will be to quantify
waterborne microbes in Missouri wetlands and relate their occurrence among the
various water bodies to measurements of water quality and land use practices
within the watershed. This will expand our studies beyond eutrophication to
include basic information on public health. We will also use collected data
to determine whether the presence (abundance) of Escherichia coli is an effective
water quality index. E. coli has long been used as an indicator of fecal contamination
(Whitman et al., 1995), but its use in predicting the presence of pathogenic
protozoans in water supplies is not clear. By including this organism in our
monitoring program, we can determine whether the presence of the fecal E. coli
correlates with the presence of Giardia cysts and/or Cryptosporidium oocysts.
We will test the merit of using E. coli as a surrogate indicator for these pathogens.

Our molecular biomonitoring program will focus on the Eagle Bluffs Conservation
Area in mid-Missouri, including the adjacent wetland treatment system for city
wastewater. This large area (> 4000 acres) is located 6 miles southeast of Columbia,
Missouri, in the flood plain of the Missouri River. Water flows from the city
treatment system into the conservation area wetlands. Water samples will be
collected weekly at multiple sites in the treatment and conservation wetlands,
and samples will be assayed for microorganisms and other aspects of water quality
as described above. The proposed project is multidiciplinary, combining the
respective expertise of three principal investigators. Dr. J. Jones (UM, Natural
Resources) will be responsible for the limnological aspects of the project,
and Drs. J. Erickson (UM, Biochemistry and Plant Pathology) and M. Milanick
(UM, Medical Physiology) will be responsible for the molecular assays for pathogen
detection and quantitation.

In summary, during the 12 months of this proposed project we will collect
water samples from conservation wetlands and the adjacent wetland treatment
system, and measure a variety of water quality parameters, as well as determine
the incidence of total coliforms, fecal coliforms, E. coli, and the protozoan
pathogens Giardia and Cryptosporidium. These data will allow us to determine
the quality of water in the wetlands, the correlation between microbiological
and standard limnological parameters, and the efficacy of using fecal E. coli
as an indicator of the protozoan pathogens. Ultimately, we expect to incorporate
the techniques developed in this project into our studies of water quality in
Missouri resevoirs. A review of the literature suggests this expanded effort
will be one of the first large-scale, comparative lake studies to include measurement
of waterborne pathogens in a routine, water quality monitoring.